The present invention relates to carboxamide-substituted dyes, the production and use of such dyes as labeling groups in analytics.
Owing to their very good spectral properties, dyes of the class of xanthene dyes and triphenylmethane dyes and also related derivatives are the preferred labeling groups used in chemical, biological and medical analytics (J. Slavik, Fluorescent Probes in Cellular and Molecular Biology, CRC Press, Boca Raton, Ann Arbor, London, Tokyo, 1994). WO 00/64986 and WO 00/64987 describe dyes of the related classes of carbopyronines and amidopyrylium dyes. In this connection, dyes having a very high fluorescence quantum yield are especially important, since fluorescence enables the labeled analyte to be detected with very high sensitivity. However, nonfluorescent derivatives also gain increasing importance as quenchers in special processes.
The use as labeling groups in processes for detecting analytes requires, aside from simple and reliable detectability, good solubility in various solvents, in particular in aqueous systems. Furthermore, compounds of this kind should be simple and inexpensive to produce and have high stability and good storability.
Many dyes of the abovementioned classes of dye possess a carboxyl group which causes the formation of a colorless lactone, owing to its position and the molecular structure as a function of the solvent environment and the pH, for example even in a neutral or weakly basic solution. In this context, cf. K. H. Drexhage, Structure and Properties of Laser Dyes, in: F. P. Schäfer, Topics in Applied Physics, Vol. 1, Dye Lasers, Springer-Verlag, Berlin, Heidelberg, N.Y., 1973, for example.
The usual covalent coupling, for example via an active ester, of a carboxyl group of this type to the primary amino group of an analyte (peptide etc.) produces an acid amide which rearranges immediately according to the following diagram to give a lactam:

Said lactam, however, is colorless under physiological conditions, thus rendering labeling with the dye unsuccessful, i.e. the labeled analyte cannot be detected by way of absorption and/or fluorescence of the labeling group. Therefore, many known and readily accessible dyes (e.g. rhodamines) are ruled out regarding the use as marker dyes. Previously, this difficulty has been avoided only by introducing additional coupling groups into the dye molecule. However, the precursors required for this are usually difficult to access and require complicated synthesis steps.
WO 02/055512 discloses the preparation of amide derivatives from fluorescein dyes, which comprises first converting the carboxylic acid group at elevated temperatures to give an active ester and then reacting said active ester with a secondary amine in an aqueous solvent mixture. The process according to WO 02/055512 is only applicable to fluorescein and its derivatives and cannot be transferred to other classes of dye. The disclosed process conditions such as temperature and solvents used are in particular not applicable to amino or/and imino group-containing compounds such as, for example, rhodamines. Firstly, the temperatures set for active ester formation result in by- and degradation products, and secondly, the water used as solvent in the reaction of the active ester with an amine can react with said active ester and this in turn results in undesired products.
It is therefore an object of the present invention to modify in a simple manner lactone- or lactam-forming dyes with different functional groups in such a way that they may be used as markers in various fields.